【生物课件】第七章 色谱技术 [Chapter 7 Chromatography]

1、本章主要知识点本章主要知识点cqKd迁移距离流动相在色谱系统中的溶质的迁移距离fRsdmmfAKAARsdmeVKVV22/1)(54. 5WtNR2)(16bRWtN NLH HPLCHPLC ChromatogramSource RPC30m15m5mInfluence of Particle Size on the Separation effect常用的离子交换树脂Mini Q PC 3.2/3: Glutathione synthetaseSDS-PAGEStarting materialPeak 2Expanded Bed分子筛凝胶色谱分子筛凝胶色谱原理Desalting prot

2、einsproteinssalts疏水作用层析(Hydrophobic Interaction Chromatography）Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to their hydrophobicityMechanism of HICA 2800.04 AUNa2SO41.0 M疏水层析操作疏水层析操作凝胶色谱填料亲和色谱（Affinity chromatography）AC ver 9903263

3、0Steric considerations &spacer armsSmall ligand (1,000)Risk of steric interference with binding between matrix and target moleculeOften need spacer armbut watch out for adsorption to the spacer!Spacer armCompetitive elution蛋白质分离常用的色谱方法AIEXTechniqueUltrafiltrationUFSuperdex 200 p.g.GFPurification

4、factorQ Sepharose FF 10 times purification 82% yieldPhenyl Sepharose HPHICY.-F. Liao et al. (1996)J. Biol. Chem. 271, 2834863622719Purification of recombinant a-mannosidase from Pichia pastorisKTA explorer 100色谱系统色谱系统KTA explorer 100色谱系统工作流程色谱系统工作流程纸层析纸层析展开剂的选择展开剂的选择薄层色谱法薄层色谱法多肽的分离纯化多肽的分离纯化5 / S. Re

5、nlund: Peptides from all sources/ ver. 3.1, 980610Separation techniquesPeptideProteinRPCGFIEXIEXGFRPCPeptides from All SourcesPeptides from All SourcesHow does a peptide differ from a protein?12 / S. Renlund: Peptides from all sources/ ver. 3.1, 980610Synthetic PeptidesSynthetic PeptidesRPC method o

6、ptimisation Make test runsat two pH valuespH 2.5Make gradient optimisation at both pH valuesScale up and purifyIdentification:MS, A A SequencePurity check: RP-HPLC, IEX, CZE pH 7.06Strategy/AC/1998/JB/. DanielssonProtein PurificationProtein PurificationAnalytical toolsA rapid and reliable assay for

7、the target proteinPurity determination(e.g. SDS-PAGE)Total protein determination (e.g. colorimetric method)蛋白的纯化蛋白的纯化11Strategy/AC/1998/JB/. DanielssonThree Phase Strategy - Ranking of Chromatography TechniquesThree Phase Strategy - Ranking of Chromatography TechniquesTechniqueCaptureIntermediatePol

8、ishingGFIEXHICACRPCConsiderationsLimited sample volumeLimited flow rate rangeProtein ligand is sensitiveto harsh cleaning conditionsUse of organic solvents,loss of biological activityTechniquePptHICAIEXCIEXACPurificationfactor720240818647Porcine cerebella homogenateRESOURCE QAmmonium sulfateprecipit

9、ationButyl Sepharose Fast FlowRESOURCE sHiTrap HeparinG Protein Receptor Kinase PurificationRec a-Mannosidase Purification from PichiaTechniquePurificationfactor63622719UFGFAIEXHICPhenyl Sepharose HPQ Sepharose FFSuperdex 200 pgUltrafiltrationDNA Binding Protein PurificationTechniqueDNA-1 Sepharose CIEX